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Mutations of SYCP3 are rare in infertile Spanish men with meiotic arrest
Mutations of SYCP3 are rare in infertile Spanish men with meiotic arrest No functional SYCP3 exonic SNP was found in infertile Spanish patients with meiosis arrest, suggesting that SYCP3 mutations very likely are not relevant in Spain in genetic susceptibility to meiosis arrest, just as in other studied European populations. (Fertil Steril 2007;88:988–9. 2007 by American Society for Reproductive Medicine.)
The SYCP3 gene, located on chromosome 12, encodes a DNA-binding protein as a component of the synaptonemal complex that regulates the synapsis between homologous chromosomes during meiosis of germ cells (1). Although the SYCP3 involvement in male fertility has been well established by knockout mice (1, 2), the role of the presence of SYCP3 variants in male infertility as a result of meiotic arrest is still a matter of debate (3, 4). The goal of our study was to evaluate the frequency of SYCP3 mutations in our population to better define the relationship between SYCP3 variants and defects of meiotic process that lead to inefficient spermatogenesis. Twenty-two infertile men with a phenotype of azoospermia (n ¼ 14) or severe oligozoospermia (<5 million sperm per mL; n ¼ 8) who underwent testicular biopsy showed a histological pattern of a complete or incomplete maturation arrest of spermatogenesis in primary spermatocyte and were subsequently screened for sequence variants in SYCP3 gene. Men with a chromosomal aberration or with a Y-chromosome microdeletion had been previously excluded from the study. Patients were recruited from the Andrology Unit of the Fundaci o Puigvert (Barcelona, Spain) and gave their informed consent for the study, which was approved by the institutional ethical committee. All of them were Caucasians of Spanish (n ¼ 20) and Maghribian (n ¼ 2) origin. SYCP3-coding and immediately flanking regions were amplified in seven fragments (primer sequences are described in Table 1) that were analyzed by single-strand conformation polymorphism (SSCP)-heteroduplex technique
Received July 14, 2006; revised and accepted November 30, 2006. n Sanitaria/Fondos Supported by grants from Fondo de Investigacio Europeos de Desarrollo Regional (PI02/0120, PI05/0759), Red de tica Clınica y Molecular ISCiii (C03/07), the GenerCentros de Gene alitat de Catalunya (2005SGR00018), and the Institut Catala` de la n Sanitaria Salut. S.L. is supported by Fondo de Investigacio (CP03/00088). Reprint requests: Sara Larriba, Ph.D., Medical and Molecular Genetics IDIBELL (Institut d’Investigacio de Bellvitge), Gran Center, Fundacio Vıa s/n km. 2.7, 08907 L’Hospitalet de Llobregat, Barcelona, Spain (FAX: 34-93-260-74-14; E-mail: [email protected]).
988
(Genephor Electrophoresis Unit; Amersham-Pharmacia Biotech, Buckinghamshire, United Kingdom) using 12.5% polyacrylamide gels (ExcelGel, Amersham Biosciences, Uppsala, Sweden). The characterization of sequence alterations was performed by directly sequencing a new PCR product corresponding to the fragments displaying mobility shift. We found only one azoospermic Spanish individual who was heterozygous for the noncoding c.454-9_13delTTTAT variant. To check whether this variant affects splicing, the exon-skipping analysis on lymphocyte complementary DNA samples was performed and evidenced no difference in exon splicing among individuals who were either presenting these variants or not (data not shown). No change was observed in the coding regions, so that amino acid sequence remained unchanged. Our results add information about SYCP3 mutational studies in patients from different populations who have infertility caused by maturation arrest of spermatogenesis. An association was previously observed in North American subjects: 2 of 19 azoospermic patients, one Hispanic and the other Arab, presented a c.643delA mutation causing defective SYCP3 expression (3); however, this was not present in European populations, either in 36 azoospermic and 22 severe oligozoospermic patients from Belgium (4) or in our study on a Mediterranean population, suggesting that the cause of this discordance may be related to the influence of ethnicity differences. We conclude that SYCP3 mutations very likely are not relevant in genetic susceptibility to meiosis arrest in European individuals and suggest that genetic background could explain the differences observed in the frequency of the SYCP3 genetic defects from different populations. Because of the multiple components that participate in meiotic chromosome pairing and synapsis (cohesin complex subunits, recombination proteins, and other synaptonemal complex subunits apart from SYCP3) (5), genetic alterations in one or several of these subunits could be responsible for meiosis arrest. Acknowledgments: The authors are indebted to the patients who participated in this study.
Fertility and Sterility Vol. 88, No. 4, October 2007 Copyright ª2007 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2006.11.163
TABLE 1 Oligonucleotide primers used in mutation analysis. Parameter
Primer
Exon 2a SYCP3EF1-1 SYCP3EF1-2 SYCP3ER1-1 Exon 3–4 SYCP3E2,3-F SYCP3E2,3-R Exon 5 SYCP3E4-F SYCP3E4-R Exon 6 SYCP3E5-F SYCP3E5-R Exon 7 SYCP3E6-F SYCP3E6-R Exon 8 SYCP3E7F SYCP3E7R Exon 9 SYCP3E8F SYCP3E8R a
50 -gtgagctaccgtgcttggcc-30 50 -cttcttaacttttggccccc-30 50 -aaggacatcactgcccaac-30 50 -cctaaaaatccccaaatgaag-30 50 -acaagtatctcctaaaaccta-30 50 -accaaccttacccatatttact-30 50 -agccaccgtgccgatcct-30 50 -acagggcttttcttctattagt-30 50 -attcctacttgagacttggttg-30 50 -atctaaatgtggtcataatagtt-30 50 -tatcacttaaatcagaaacaatg-30 50 -agtaggtattttggtattcatgt-30 50 -atctcttgctgctgctgtttc-30 50 -ttgctgaaataggtataacactt-30 50 -gaggtcttacaatgaaacaggt-30
Primer sequences for exon 2 have been described elsewhere (3).
Martınez. SYCP3 gene and spermatogenic arrest. Fertil Steril 2007.
Juanjo Martınez, B.S.a Sandra Bonache, B.S.a Alejandro Carvajal, M.D.b Lluıs Bassas, M.D., Ph.D.b Sara Larriba, Ph.D.a a Medical and Molecular Genetics Center–Fundaci o IDIBELL, L’Hospitalet de Llobregat; and b Andrology Service, Fundaci o Puigvert, Barcelona, Spain REFERENCES 1. Yuan L, Liu JG, Zhao J, Brundell E, Daneholt B, Hoog C. The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility. Mol Cell 2000;5:73–83.
Fertility and Sterility
2. Kolas NK, Yuan L, Hoog C, Heng HH, Marcon E, Moens PB. Male mouse meiotic chromosome cores deficient in structural proteins SYCP3 and SYCP2 align by homology but fail to synapse and have possible impaired specificity of chromatin loop attachment. Cytogenet Genome Res 2004;105:182–8. 3. Miyamoto T, Hasuike S, Yogev L, Maduro MR, Ishikawa M, Westphal H, et al. Azoospermia in patients heterozygous for a mutation in SYCP3. Lancet 2003;362:1714–9. 4. Stouffs K, Lissens W, Tournaye H, Van Steirteghem A, Liebaers I. SYCP3 mutations are uncommon in patients with azoospermia. Fertil Steril 2005;84: 1019–20. 5. Pelttari J, Hoja MR, Yuan L, Liu JG, Brundell E, Moens P, et al. A meiotic chromosomal core consisting of cohesin complex proteins recruits DNA recombination proteins and promotes synapsis in the absence of an axial element in mammalian meiotic cells. Mol Cell Biol 2001;21: 5667–77.
989
The SYCP3 gene, located on chromosome 12, encodes a DNA-binding protein as a component of the synaptonemal complex that regulates the synapsis between homologous chromosomes during meiosis of germ cells (1). Although the SYCP3 involvement in male fertility has been well established by knockout mice (1, 2), the role of the presence of SYCP3 variants in male infertility as a result of meiotic arrest is still a matter of debate (3, 4). The goal of our study was to evaluate the frequency of SYCP3 mutations in our population to better define the relationship between SYCP3 variants and defects of meiotic process that lead to inefficient spermatogenesis. Twenty-two infertile men with a phenotype of azoospermia (n ¼ 14) or severe oligozoospermia (<5 million sperm per mL; n ¼ 8) who underwent testicular biopsy showed a histological pattern of a complete or incomplete maturation arrest of spermatogenesis in primary spermatocyte and were subsequently screened for sequence variants in SYCP3 gene. Men with a chromosomal aberration or with a Y-chromosome microdeletion had been previously excluded from the study. Patients were recruited from the Andrology Unit of the Fundaci o Puigvert (Barcelona, Spain) and gave their informed consent for the study, which was approved by the institutional ethical committee. All of them were Caucasians of Spanish (n ¼ 20) and Maghribian (n ¼ 2) origin. SYCP3-coding and immediately flanking regions were amplified in seven fragments (primer sequences are described in Table 1) that were analyzed by single-strand conformation polymorphism (SSCP)-heteroduplex technique
Received July 14, 2006; revised and accepted November 30, 2006. n Sanitaria/Fondos Supported by grants from Fondo de Investigacio Europeos de Desarrollo Regional (PI02/0120, PI05/0759), Red de tica Clınica y Molecular ISCiii (C03/07), the GenerCentros de Gene alitat de Catalunya (2005SGR00018), and the Institut Catala` de la n Sanitaria Salut. S.L. is supported by Fondo de Investigacio (CP03/00088). Reprint requests: Sara Larriba, Ph.D., Medical and Molecular Genetics IDIBELL (Institut d’Investigacio de Bellvitge), Gran Center, Fundacio Vıa s/n km. 2.7, 08907 L’Hospitalet de Llobregat, Barcelona, Spain (FAX: 34-93-260-74-14; E-mail: [email protected]).
988
(Genephor Electrophoresis Unit; Amersham-Pharmacia Biotech, Buckinghamshire, United Kingdom) using 12.5% polyacrylamide gels (ExcelGel, Amersham Biosciences, Uppsala, Sweden). The characterization of sequence alterations was performed by directly sequencing a new PCR product corresponding to the fragments displaying mobility shift. We found only one azoospermic Spanish individual who was heterozygous for the noncoding c.454-9_13delTTTAT variant. To check whether this variant affects splicing, the exon-skipping analysis on lymphocyte complementary DNA samples was performed and evidenced no difference in exon splicing among individuals who were either presenting these variants or not (data not shown). No change was observed in the coding regions, so that amino acid sequence remained unchanged. Our results add information about SYCP3 mutational studies in patients from different populations who have infertility caused by maturation arrest of spermatogenesis. An association was previously observed in North American subjects: 2 of 19 azoospermic patients, one Hispanic and the other Arab, presented a c.643delA mutation causing defective SYCP3 expression (3); however, this was not present in European populations, either in 36 azoospermic and 22 severe oligozoospermic patients from Belgium (4) or in our study on a Mediterranean population, suggesting that the cause of this discordance may be related to the influence of ethnicity differences. We conclude that SYCP3 mutations very likely are not relevant in genetic susceptibility to meiosis arrest in European individuals and suggest that genetic background could explain the differences observed in the frequency of the SYCP3 genetic defects from different populations. Because of the multiple components that participate in meiotic chromosome pairing and synapsis (cohesin complex subunits, recombination proteins, and other synaptonemal complex subunits apart from SYCP3) (5), genetic alterations in one or several of these subunits could be responsible for meiosis arrest. Acknowledgments: The authors are indebted to the patients who participated in this study.
Fertility and Sterility Vol. 88, No. 4, October 2007 Copyright ª2007 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2006.11.163
TABLE 1 Oligonucleotide primers used in mutation analysis. Parameter
Primer
Exon 2a SYCP3EF1-1 SYCP3EF1-2 SYCP3ER1-1 Exon 3–4 SYCP3E2,3-F SYCP3E2,3-R Exon 5 SYCP3E4-F SYCP3E4-R Exon 6 SYCP3E5-F SYCP3E5-R Exon 7 SYCP3E6-F SYCP3E6-R Exon 8 SYCP3E7F SYCP3E7R Exon 9 SYCP3E8F SYCP3E8R a
50 -gtgagctaccgtgcttggcc-30 50 -cttcttaacttttggccccc-30 50 -aaggacatcactgcccaac-30 50 -cctaaaaatccccaaatgaag-30 50 -acaagtatctcctaaaaccta-30 50 -accaaccttacccatatttact-30 50 -agccaccgtgccgatcct-30 50 -acagggcttttcttctattagt-30 50 -attcctacttgagacttggttg-30 50 -atctaaatgtggtcataatagtt-30 50 -tatcacttaaatcagaaacaatg-30 50 -agtaggtattttggtattcatgt-30 50 -atctcttgctgctgctgtttc-30 50 -ttgctgaaataggtataacactt-30 50 -gaggtcttacaatgaaacaggt-30
Primer sequences for exon 2 have been described elsewhere (3).
Martınez. SYCP3 gene and spermatogenic arrest. Fertil Steril 2007.
Juanjo Martınez, B.S.a Sandra Bonache, B.S.a Alejandro Carvajal, M.D.b Lluıs Bassas, M.D., Ph.D.b Sara Larriba, Ph.D.a a Medical and Molecular Genetics Center–Fundaci o IDIBELL, L’Hospitalet de Llobregat; and b Andrology Service, Fundaci o Puigvert, Barcelona, Spain REFERENCES 1. Yuan L, Liu JG, Zhao J, Brundell E, Daneholt B, Hoog C. The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility. Mol Cell 2000;5:73–83.
Fertility and Sterility
2. Kolas NK, Yuan L, Hoog C, Heng HH, Marcon E, Moens PB. Male mouse meiotic chromosome cores deficient in structural proteins SYCP3 and SYCP2 align by homology but fail to synapse and have possible impaired specificity of chromatin loop attachment. Cytogenet Genome Res 2004;105:182–8. 3. Miyamoto T, Hasuike S, Yogev L, Maduro MR, Ishikawa M, Westphal H, et al. Azoospermia in patients heterozygous for a mutation in SYCP3. Lancet 2003;362:1714–9. 4. Stouffs K, Lissens W, Tournaye H, Van Steirteghem A, Liebaers I. SYCP3 mutations are uncommon in patients with azoospermia. Fertil Steril 2005;84: 1019–20. 5. Pelttari J, Hoja MR, Yuan L, Liu JG, Brundell E, Moens P, et al. A meiotic chromosomal core consisting of cohesin complex proteins recruits DNA recombination proteins and promotes synapsis in the absence of an axial element in mammalian meiotic cells. Mol Cell Biol 2001;21: 5667–77.
989